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  • Title: Determination of beta1,4-galactosyltransferase enzymatic activity by capillary electrophoresis and laser-induced fluorescence detection.
    Author: Snow DM, Shaper JH, Shaper NL, Hart GW.
    Journal: Anal Biochem; 1999 Jun 15; 271(1):36-42. PubMed ID: 10361002.
    Abstract:
    We have developed a nonradioactive method to assay UDP-Gal:beta-d-GlcNAcbeta1,4-galactosyltransferase (beta4GalT-I) enzymatic activity. Capillary electrophoresis combined with laser-induced fluorescence detection (CE-LIF) was employed to provide a baseline separation of FITC-conjugated O-GlcNAc-containing substrate peptides and galactose-capped product peptides, while at the same time allowing a level of detection in the low attomole range (10(-18)). The addition of 2 mM hexamethylene diamine to the borate-based capillary electrophoretic buffer modulated the electroosmotic flow, resulting in optimum separation of the glycopeptide product from reactant. beta4GalT-I activity was dependent upon the addition of both manganese and UDP-galactose. Using this assay, we show that two beta4GalT-I constructs, predicted to localize to different intracellular compartments, are enzymatically active when expressed in vitro using a rabbit reticulocyte transcription-translation system. The high sensitivity of product detection by CE-LIF in combination with in vitro transcription-translation is applicable to the facile determination of the enzymatic activity of other newly cloned glycosyltransferases.
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